In vivo control of Toxoplasma gondii by zebrafish macrophages

Toxoplasma gondii is an obligate intracellular parasite capable of invading any nucleated cell. Three main clonal lineages (type I, II, III) exist and murine models have driven the understanding of general and strain-specific immune mechanisms underlying Toxoplasma infection. However, murine models are limited for studying parasite-leukocyte interactions in vivo, and discrepancies exist between cellular immune responses observed in mouse versus human cells. Here, we develop a zebrafish infection model to study the innate immune response to Toxoplasma in vivo. By infecting the zebrafish hindbrain ventricle, and using high-resolution microscopy techniques coupled with computer vision driven automated image analysis, we reveal that Toxoplasma invades and replicates inside a parasitophorous vacuole to which type I and III parasites recruit host cell mitochondria. We show that type II and III strains maintain a higher infectious burden than type I strains. To understand how parasites are being cleared in vivo, we analyzed Toxoplasma-macrophage interactions using time-lapse and correlative light and electron microscopy. Strikingly, macrophages are recruited to the infection site and play a key role in Toxoplasma control. These results highlight in vivo control of Toxoplasma by macrophages, and illuminate the possibility to exploit zebrafish for discoveries within the field of parasite immunity.


Introduction
Toxoplasma gondii is a successful human pathogen that often remains asymptomatic, however complications arise in the immunocompromised and in neonates if infection is contracted during pregnancy (1). Toxoplasma exist as invasive rapidly replicating tachyzoites in intermediate hosts (such as rodents and livestock), and convert into bradyzoite cysts in immune privileged sites and long-lived cells (such as the brain and muscle tissue) during chronic infection (2). Once inside the host cell, parasites reside in a non-fusogenic parasitophorous vacuole (PV) where Toxoplasma asexually replicates (3). Egress leads to dissemination into neighboring tissues, culminating in systemic infection. Predation of intermediate hosts by the definitive feline host completes the Toxoplasma life cycle. Control of infection by the host immune response is thus critical for both host survival and for contin-ued parasite transmission. As a result of its well-understood life cycle, Toxoplasma has emerged as a valuable model organism to understand the balance of pathogen survival and innate cellular immune control. Three clonal lineages of Toxoplasma dominate across Europe and South America, namely the type I, II and III strains (4). These three closely-related Toxoplasma strains have been characterized by the severity of infections they cause in murine models (5). Infection with type I parasites causes acute mouse mortality, whereas infection with type II and type III parasites progress towards chronic infection (6,7). In humans, it is thought that type II strains predominate in Europe, yet strain-dependent differences in pathogenesis and host responses are poorly understood (8,9). Innate immune mechanisms against Toxoplasma infection have been studied in vitro using both murine and human cell lines and in vivo using mice. In vivo studies have shown monocytes and neutrophils are recruited to the intestine upon oral infection, and are the major cell types infected with Toxoplasma both in vivo and ex vivo in human peripheral blood (10)(11)(12)(13). The importance of neutrophils in parasite control in vivo is not fully understood, yet neutrophilspecific depletion studies have suggested a minor protective role against Toxoplasma (14,15). In contrast, inflammatory monocytes are the first responders to infection and are crucial for controlling acute Toxoplasma infection (16)(17)(18). Pioneering work identified the ability of macrophages to kill Toxoplasma (19,20), by employing both IFN-γ-dependent and -independent mechanisms to control intracellular parasite replication (21)(22)(23). While the mouse is a natural intermediate host and remains an important model to understand Toxoplasma pathogenesis, differences are emerging between the mouse and human in mechanisms of parasite control (5,(24)(25)(26)(27)(28). Therefore, to complement in vivo murine studies, a novel animal model can benefit analysis of Toxoplasma control on a cellular and molecular level. Zebrafish are a well-established model for studying infection and immunity (29)(30)(31)(32). Coupled with their optical accessibility during early development, zebrafish larvae are highly suited for non-invasive study of Toxoplasma infection and host response in real-time in vivo (31,32). Here we develop a zebrafish infection model to study straindependent infectivity and leukocyte response to Toxoplasma infection. We discover type II (Pru) and III (CEP) parasites maintain a higher infectious burden than type I (RH) parasites. We show macrophages are crucial in the clearance of viable parasites. Our zebrafish infection model can be used as a novel platform to enable unprecedented discoveries in strain-dependent parasite immunity.

Results
Intracellular Toxoplasma replicate in the zebrafish hindbrain ventricle. To develop a Toxoplasma-zebrafish infection model, we tested if tachyzoites could replicate in zebrafish larvae. We first used Toxoplasma type I (RH) strain, since it is known to grow faster in vitro and survive longer extracellularly than type II (Pru) and type III (CEP) strains (6,33,34). We injected zebrafish larvae 3 days post-fertilization (dpf) in the hindbrain ventricle (HBV) with~5x10 3 type I strain tachyzoites expressing GFP and followed infection for 24 hours at 33°C (Sup. Fig. 1A). We observed parasite replication in vivo using time-lapse widefield fluorescent microscopy (Sup. Fig. 1B, Movie 1). Consistent with this, confocal microscopy shows the percentage of vacuoles containing two or more tachyzoites significantly increasing with time ( Fig. 1A and B). To identify the intracellular location of replicating parasites, infected larvae were fixed and stained for granule antigen 2 (GRA2), a dense granule protein that accumulates in the PV lumen (35). Here, GRA2 accumulates around single and replicating parasites, highlighting PV formation in vivo (Fig. 1C). To investigate parasite morphology and location at 6hpi, 3D correlative light and electron microscopy (CLEM) was performed on the HBV of infected zebrafish. In this case, we observe that parasites are inside zebrafish cells and display host mitochondrial association (Fig. 1D, other text Sup. Fig.  1C, Movie 2), a hallmark of intracellular type I parasites previously observed in mouse and human cells (36). Moreover, tachyzoites can be observed as singlets, replicating doublets (thus joined together) or fully replicated doublets (with a distinct membrane around each tachyzoite) (Fig. 1D, Sup. Type II and III parasites are more efficient than type I parasites at establishing infection in vivo. To determine if parasite strain can affect parasite burden and host response in our zebrafish model, we infected larvae with~5x10 3 type I, type II or type III Toxoplasma-GFP (Sup. Fig. 2A). In all cases, infected larvae show 100% survival and no adverse effects up to 48hpi (Sup. Fig. 2B). Analysis by fluorescent stereomicroscopy showed, from initial parasite input, parasite burden is reduced~95% by 6hpi, suggesting~5% of parasites successfully invade zebrafish cells and establish infection. To quantify parasite burden in a high-throughput manner we optimized an automated quantification pipeline using ZedMate (37) for the different strain types at 6 and 24hpi. Strikingly, type II and III parasite burden is~3x higher than type I parasite burden at 6hpi ( Fig. 2A and B, Sup. Fig. 2C). However, once established at 6hpi, all 3 strain types persist equally and decrease by~20% between 6 and 24hpi. To test if host mitochondrial association is observed across the three strain types, we stained host mitochondria in the HBV of infected zebrafish larvae. In agreement with in vitro observations (36), both type I and type III parasites (and not type II parasites) show clear host mitochondrial association (Fig. 2C). These results demonstrate that strain typedependent host mitochondrial association characteristics are conserved in zebrafish in vivo.

Macrophage and neutrophil response to parasite infection in vivo.
To analyze Toxoplasmamacrophage interactions over time, 3dpf transgenic larvae possessing red macrophages Tg(mpeg1:Gal4-FF) gl25 /Tg(UAS-E1b:nfsB.mCherry) c264 (herein referred to as mpeg1:G/U:mCherry), were infected with type I, II or III Toxoplasma-GFP, and macrophage recruitment was quantified by fluorescent stereomicroscopy. As compared to mock injection, the number of macrophages recruited to the infection site is significantly increased (~1.5 fold) for all three strain types at both 6 and 24hpi (Fig. 3A). To analyze Toxoplasma-neutrophil interactions over time, 3dpf transgenic larvae possessing red neutrophils Tg(lyz:dsRed) nz50 (herein referred to as lyz:dsRed), were infected with type I, II or III Toxoplasma-GFP, and neutrophil recruitment was quantified by fluorescent stereomicroscopy. Here, the number of neutrophils recruited to the infection site is significantly increased (~3 fold) as compared to mock injection for all three strain types at 6hpi (Fig. 3B). In contrast to macrophages, which remain at the infection site by 24hpi, the number of neutrophils recruited to the infection site (for all three strain types) is significantly decreased to basal levels by 24hpi.

Macrophages control parasite burden in vivo.
To analyze the interactions between type I Toxoplasma and macrophages in depth, we imaged infected mpeg1:G/U:mCherry larvae with Toxoplasma-GFP at 6hpi using confocal microscopy and 3D CLEM. In this case, the majority of intact type I parasites contained within macrophages are single tachyzoites inside PVs (as judged by host mitochondria association to the membrane surrounding the parasites) (Sup. Fig. 3A). This suggests that macrophages may prevent parasite replication. To follow the fate of type I parasites engulfed by macrophages in real-time, mpeg1:G/U:mCherry larvae infected with Toxoplasma-GFP were imaged by time-lapse confocal microscopy. In this case, we frequently observed the engulfment of parasites by macrophages followed by loss of GFP fluorescence, suggesting active parasite degradation (Fig. 4A, Movie 3). Consistent with this, 3D CLEM showed parasite degradation inside macrophages, as identified by fragmentation of tachyzoite organelles (Fig. 4B, Sup. Fig. 3B). To test the role of macrophages in Toxoplasma infection in vivo, mpeg1:G/U:mCherry larvae were pre-treated with control (DMSO) or metronidazole (Mtz) to ablate macrophages (Sup. Fig. 3C and D). In the absence of macrophages, infected larvae showed 100% survival (Sup. Fig. 3E). However, parasite burden is significantly increased, suggesting macrophages are responsible for parasite clearance in vivo ( Fig. 4C and D, Sup. Fig. 3F). Similar results are observed with type II and III strain infection of macrophage-ablated larvae (Sup. Fig. 3G). To analyze the viability of parasites that are cleared by macrophages, we performed vacuole volume quantification and show that DMSO and Mtz-treated larvae are comprised of equally replicating Toxoplasma tachyzoites (Fig. 4E). These data suggest that macrophages have a dominant role in clearing healthy viable parasites rather than supporting the parasite's replicative niche.

Discussion
Zebrafish infection models for studying eukaryotic parasites and other human pathogens are beginning to emerge (31,32,38). In this study, we establish a novel Toxoplasma infection model using zebrafish larvae to explore host-parasite interaction in vivo. We find the three main clonal lineages of Toxoplasma are able to invade zebrafish cells and replicate within their PV, and reveal that macrophages are key in controlling viable parasites in vivo. Using confocal microscopy and CLEM, we visualize single and replicating type I tachyzoites in the zebrafish HBV exhibiting host mitochondrial association. The relatively slow replication cycle of Toxoplasma observed in tissue culture cells in vitro (>6h) is consistent with what we observe in the zebrafish HBV. GRA2 staining and CLEM of replicating tachyzoites strongly suggests PV formation and is indicative of normal type I parasite behavior as demonstrated in vitro using tissue culture cells and in vivo using other animal models (39). The zebrafish HBV is well established to investigate host response to infection (30)(31)(32). We do not observe Toxoplasma dissemination from the HBV and this allows us to monitor leukocyte-parasite interactions in a localized area. Here, type II and III strains are more efficient than type I strains at maintaining a higher infectious burden. This suggests type II and III strains may be more efficient at invading non-phagocytic cell types found in the HBV and/or evading clearance by host cells. Together with CLEM evidence we conclude that Toxoplasma favor replication within non-phagocytic cells (such as epithelial and neuronal cells) within the HBV of zebrafish larvae. Both live-cell imaging and CLEM showed type I parasite uptake and clearance by macrophages. In all cases of macrophage-parasite interaction captured by CLEM, macrophages retained their fluorescence during Toxoplasma infection and had intact nuclei and mitochondria, indicative of a healthy host cell. Our evidence obtained from time-lapse microscopy, fixed 3D CLEM and macrophage ablation highlights active parasite clearance by zebrafish macrophages in vivo occurs within the first 6hpi. Therefore, future work using this zebrafish infection model could uniquely explore the precise anti-parasitic mechanisms employed by macrophages during Toxoplasma infection. Both live-cell imaging and CLEM showed type I parasite uptake and clearance by macrophages. In all cases of macrophage-parasite interaction captured by CLEM, macrophages retained their fluorescence during Toxoplasma infection and had intact nuclei and mitochondria, indicative of a healthy host cell. Our evidence obtained from time-lapse microscopy, fixed 3D CLEM and macrophage ablation highlights active parasite clearance by zebrafish macrophages in vivo occurs within the first 6hpi. Therefore, future work using this zebrafish infection model could uniquely explore the precise anti-parasitic mechanisms employed by macrophages during Toxoplasma infection. In murine in vivo models, various leukocytes have been implicated in trafficking Toxoplasma from the site of infection. Examples include infected neutrophils that pass from the intestine to the lumen (11), as well as infected macrophages and dendritic cells that pass the blood brain barrier (40). In light of this, it is intriguing to note that our study using time-lapse microscopy shows a minority of parasite-infected macrophages moving through the brain tissue for possible parasite transport (Sup. Fig. 4A). Careful analysis of our 3D CLEM data also identified an actively replicating type I parasite inside a macrophage exhibiting host mitochondrial association to the PV (Sup. Fig. 4B-C, Movie 4). This obser-vation is reminiscent of parasite replication "hot spots" described in vivo in the murine intestinal villi (11) . Overall, it is remarkable that zebrafish macrophages during Toxoplasma infection in vivo have the capacity to phenocopy known behavior exhibited by murine macrophages during Toxoplasma infection in vivo. This is the case for both the type of events observed (e.g. parasite killing, trafficking, sustaining replication), and their approximate in vivo frequency. In summary, we here establish a novel animal model for studying the in vivo innate immune response to Toxoplasma infection, and compare host response to the three main Toxoplasma strain types in vivo. We also discover the dominant role of macrophages in parasite clearance. Having established a zebrafish model of Toxoplasma infection, we have made available a unique in vivo infection platform for CRISPR targeting and high-throughput drug screens that, together with time-lapse microscopy, can be used to identify determinants underlying Toxoplasma infection control.

Materials and Methods
Ethics statement. Animal experiments were performed according to the Animals (Scientific Procedures) Act 1986 and approved by the Home Office (Project licenses: PPL P84A89400 and P4E664E3C). All experiments were conducted up to 5 days post-fertilization.
Zebrafish husbandry and maintenance. Fish were reared and maintained at 28.5°C on a 14hr light, 10hr dark cycle. Embryos obtained by natural spawning were maintained in 0.5x E2 media supplemented with 0.3 µg/ml methylene blue.
Larvae were anesthetized with 20 µg/ml tricaine (Sigma-Aldrich) during the injection procedures and for live in vivo imaging. All experiments were carried out on TraNac background (41) larvae to minimize obstruction of fluorescence signal by pigmentation leading to misrepresentation in parasite dose quantification.
Parasite culture, preparation and infection. Toxoplasma (RH/Pru/CEP) expressing GFP/luciferase or Tomato was maintained in vitro by serial passage on human foreskin fibroblasts (HFFs) cultures (ATCC). Cultures were grown in DMEM high glucose (Life Technologies) supplemented with 10% FBS (Life Technologies) at 37°C in 5% CO 2 . Parasites were prepared from 25G followed by 27G syringe-lysed HFF cultures in 10% FBS. Excess HFF material removed by centrifugation for 10min at 50 x g. After washing with PBS, Toxoplasma tachyzoites were resuspended at 2x10 6 tachyzoites/µl in PBS. During injection, tachyzoites were maintained at room temperature and passed through 29G myjector syringe (Terumo) to dissociate clumps and homogenize the suspension. Control infections were carried out using uninfected HFF cultures prepared as described above. 3dpf larvae were anesthetized and injected with~2.5nl of parasite suspension into the HBV. HBV injections were carried out as previously described (42). Larvae are optimally maintained at 28.5°C, but develop normally between 23-33°C (43). Toxoplasma invades and replicates at a minimum of 33°C. Infected larvae were therefore transferred into media pre-warmed to 33°C to ensure normal zebrafish development and parasite replication (Sup. Fig. 1A). Progress of infection was monitored by fluorescent stereomicroscopy (Leica M205FA, Leica Microsystems).

Quantification of parasite dose and burden.
For parasite dose quantification z-stack images of the infected hindbrain were taken within 5-10min using the Leica M205FA fluorescent stereomicroscope on a 130x magnification using a 1x objective. Images were analyzed using the particle analysis function in Fiji software (44). For manual quantification of parasite burden, z-stack images were taken using the Leica M205FA. GFP-positive punctae were quantified using the multi-point tool in Fiji software. Computer vision driven automated parasite burden quantifications were carried out using the ZedMate plugin in Fiji software to corroborate manual quantifications (37). Pixel volume quantifications were carried out by 3D projecting confocal z-stack images and using the 3D objects counter tool in Fiji (Movie 5).
Live imaging, image processing and analysis. Live in vivo imaging was performed on anesthetized larvae immobilized in 1% low melting-point agarose in 35mm glass-bottomed dishes (MatTek Corp.). Widefield microscopy was performed using a 40x objective. Z-stacks were acquired at 10-minute intervals. 60 z slices were taken at 2µm sections. Confocal microscopy was performed using the Zeiss Invert LSM 710 (Carl Zeiss AG) and the LSM 880 (Carl Zeiss AG) using a 40x and 63x objective. Z-stacks were acquired at 8minute intervals. 60 z slices were taken at 0.9µm sections per larva. For all time-lapse acquisitions, larvae were maintained at 33°C. For mitochondria staining, larvae were injected with 1nl MitoTracker® DeepRed (250µM, Life Technologies) 40min prior to embedding for live confocal microscopy.
Wholemount immunohistochemistry. Euthanized larvae were fixed overnight at 4°C in 4% paraformaldehyde supplemented with 0.4% Triton X-100 and washed in PBS, 0.4% Triton X-100 before staining. Briefly, after a 20min wash in PBS 1% Triton X-100, larvae were incubated overnight at 4°C in blocking solution: PBS, supplemented with 10% FBS, 1% DMSO and 0.1% Tween 20. Primary antibodies diluted in blocking solution were applied overnight at 4°C. Larvae were washed 4x15min with PBS supplemented with 0.1% Tween 20. Secondary antibodies diluted in blocking solution were applied overnight at 4°C. Larvae were washed 4x15min with PBS supplemented with 0.1% Tween 20. Hoechst 33342 staining of larvae was carried out at room temperature for 10min, followed by 3x10min washes with PBS, 0.1% Tween 20. Larvae were then cleared through a glycerol series before imaging by confocal microscopy (Zeiss LSM 710).

3D correlative light and electron microscopy (CLEM).
For serial blockface scanning electron microscopy (SBF SEM), euthanized larvae were fixed overnight at 4°C in 4% formaldehyde (Taab Laboratories Equipment Ltd.). Hoechst 33342 staining of larvae was carried out at room temperature for 10min without permeabilization and larvae were subsequently washed 3x10min with 0.1M phosphate buffer (PB). Larvae were embedded in 3% low-melt agarose in 35mm glass-bottomed dishes. Larvae were covered in 0.1M PB for high-resolution confocal microscopy (Zeiss LSM 710). Larvae were maintained in 1% formaldehyde in 0.1M PB until further processing. The embedded larvae were sectioned using a Leica VT1000 S vibrating blade microtome (Leica Biosystems). 50µm sections were collected and stored in 0.1M PB in a 24-well glass-bottomed plate (MatTek Corp.). The sections were imaged again using a Zeiss Invert 710 LSM confocal (Carl Zeiss AG) and a 20x Ph2 objective. The sections containing Toxoplasma were then processed following the method of the National Centre for Microscopy and Imaging Research (45). In brief they were post-fixed in 2.5% (v/v) gluteraldehyde/4% (v/v) formaldehyde in 0.1M PB for 30min at room temperature, stained in 2% osmium tetroxide/1.5% potassium ferricyanide for 1h on ice, incubated in 1% w/v thiocarbohydrazide for 20min before a second staining with 2% osmium tetroxide, and incubation overnight in 1% aqueous uranyl acetate at 4°C. Sections were stained with Walton's lead aspartate for 30min at 60°C and dehydrated stepwise through an ethanol series on ice, incubated in a 1:1 propylene oxide/Durcupan resin mixture and embedded in Durcupan ACM® resin according to the manufacturer's instructions (Sigma-Aldrich). Blocks were trimmed to a small trapezoid, excised from the resin block and attached to a SBF SEM specimen holder using conductive epoxy resin (Circuitworks CW2400). Prior to commencement of a SBF SEM imaging run, the sample was coated with a 2nm layer of platinum to further enhance conductivity using a Q150R S sputter coater (Quorun Technologies Ltd.). SBF SEM data was collected using a 3View2XP (Gatan Inc.) attached to a Sigma VP SEM (Zeiss). Inverted backscattered electron images were acquired through the entire extent of the region of interest. For each 50nm slice, a low-resolution overview image (pixel size of 50nm using a 1.5µs dwell time) and several high-resolution images of the different regions of interest (indicated magnification 5000x, pixel size of 6-7nm using a 1.5µs dwell time) were acquired. The overview image was used to relocate the region of interest defined by the confocal images of the sections. The SEM was operated in variable pressure mode at 5Pa. The 30µm aperture was used, at an accelerating voltage of 2kV. Typically, between 300 and 1000 slices were necessary for an entire region of interest. As data was collected in variable pressure mode, only minor adjustments in image alignment were needed, particularly where the field of view was altered in order to track the cell of interest. All the images were converted to tiff in Digital Micrograph (Gatan Inc.), and tiff stacks were automatically aligned using TrakEM2, a FIJI framework plug-in (Cardona et al. 2012). Manual segmentations were done in TrakEM2. For Fig. 1D, labels were exported as tiff for visualization in 3D in ClearVolume, a FIJI framework plug-in (46). For Sup. Fig. 4B, they were exported as Amira labels for visualization in 3D in Amira Software (Thermo Fisher Scientific). Movie 2 was generated in FIJI, Movie 4 in Amira, both were compressed in Quick Time Pro with the H.264 encoder.
Measurement of leukocyte recruitment to the site of infection. Anesthetized larvae were imaged 0, 6 and 24hpi by fluorescent stereomicroscopy (Leica M205FA). 15 z slices were taken at 130x magnification. Images were further analyzed using Fiji software.